Error in fread(paste0(opt$out_dir, "/zUMIs_output/", opt$project, "kept_barcodes_binned.txt"))

[shangyf-stu]

Hello! I encountered an error while running the code, but couldn't find the reason.

Here is my error message: /zUMIs_v2.9.7d/zUMIs/zUMIs.sh: line 285: 1216 Killed ${Rexc} ${zumisdir}/zUMIs-BCdetection.R ${yaml} Error in fread(paste0(opt$out_dir, "/zUMIs_output/", opt$project, "kept_barcodes_binned.txt")) : File '/dataset456/zUMIs/zUMIs_output/dataset456kept_barcodes_binned.txt' does not exist or is non-readable. getwd()=='/zUMIs' Execution halted Loading required package: yaml Loading required package: Matrix Error in gzfile(file, "rb") : cannot open the connection Calls: rds_to_loom -> readRDS -> gzfile In addition: Warning message: In gzfile(file, "rb") : cannot open compressed file '/dataset456/zUMIs/zUMIs_output/expression/dataset456.dgecounts.rds', probable reason 'No such file or directory' Execution halted Error in gzfile(file, "rb") : cannot open the connection Calls: readRDS -> gzfile In addition: Warning message: In gzfile(file, "rb") : cannot open compressed file '/dataset456/zUMIs/zUMIs_output/expression/dataset456.dgecounts.rds', probable reason 'No such file or directory' Execution halted

To Reproduce Here is my yaml file: `project: dataset456 sequence_files: file1: name: /dataset456/filter_fastp/dataset456_clean_1.fastq.gz base_definition:

• BC(1-12)
• UMI(13-20) file2: name: /dataset456/filter_fastp/dataset456_clean_2.fastq.gz base_definition:
• cDNA(1-60) reference: STAR_index: /zUMIs_v2.9.7d/star_index_2.7.1a_nogtf GTF_file: /3.2.0_star_index/Homo_sapiens.GRCh38.102.gtf exon_extension: no extension_length: 0 scaffold_length_min: 0 additional_files: additional_STAR_params: out_dir: /dataset456/zUMIs num_threads: 8 mem_limit: 20 filter_cutoffs: BC_filter: num_bases: 1 phred: 20 UMI_filter: num_bases: 1 phred: 20 barcodes: barcode_num: null barcode_file: null barcode_sharing: null automatic: yes BarcodeBinning: 1 nReadsperCell: 100 demultiplex: no counting_opts: introns: yes intronProb: no downsampling: '0' strand: 0 Ham_Dist: 0 velocyto: no primaryHit: yes multi_overlap: no fraction_overlap: 0 twoPass: yes make_stats: yes which_Stage: Filtering samtools_exec: samtools Rscript_exec: Rscript STAR_exec: STAR pigz_exec: pigz ` Thank you for your help!

[shangyf-stu]

The format of the yaml file is not convenient for viewing, I will send it again.

project: dataset456 sequence_files: file1: name: /dataset456/filter_fastp/dataset456_clean_1.fastq.gz base_definition:

• BC(1-12)
• UMI(13-20) file2: name: /dataset456/filter_fastp/dataset456_clean_2.fastq.gz base_definition:
• cDNA(1-60) reference: STAR_index: /zUMIs_v2.9.7d/star_index_2.7.1a_nogtf GTF_file: /3.2.0_star_index/Homo_sapiens.GRCh38.102.gtf exon_extension: no extension_length: 0 scaffold_length_min: 0 additional_files: additional_STAR_params: out_dir: /dataset456/zUMIs num_threads: 8 mem_limit: 20 filter_cutoffs: BC_filter: num_bases: 1 phred: 20 UMI_filter: num_bases: 1 phred: 20 barcodes: barcode_num: null barcode_file: null barcode_sharing: null automatic: yes BarcodeBinning: 1 nReadsperCell: 100 demultiplex: no counting_opts: introns: yes intronProb: no downsampling: '0' strand: 0 Ham_Dist: 0 velocyto: no primaryHit: yes multi_overlap: no fraction_overlap: 0 twoPass: yes make_stats: yes which_Stage: Filtering samtools_exec: samtools Rscript_exec: Rscript STAR_exec: STAR pigz_exec: pigz

[cziegenhain]

Seems like your job got killed for external reasons during barcode correction, could it be that you ran out of memory or similar?

[shangyf-stu]

Yes, the operation of zUMIs has been stopped due to ran out of memory. I have encountered this type of problem twice, probably including the kill error, all of which are caused by this reason. Thank you for your patient answer. Additionally, I have encountered difficulties in library that contains multiple barcodes, and I will create a new issue.